Recently it has been observed that birth rates in Teplice, a highly
polluted district in Northern Bohemia, have been reduced during periods
when sulfur dioxide levels were high. This study, which is based on data
from 2,585 parental pairs in the same region, describes an analysis of
the impact of [SO.sub.2] on fecundability in the first unprotected
menstrual cycle (FUMC). We obtained detailed personal data, including
time-to-pregnancy information, via maternal questionnaires at delivery.
We estimated individual exposures to [SO.sub.2] in each of the 4 months
before conception on the basis of continual central monitoring. Three
concentration intervals were introduced: [is less than] 40
[micro]g/[m.sup.3] (reference level); 40-80 [micro]g/[m.sup.3]; and [is
greater than or equal to] 80 [micro]g/[m.sup.3]. We estimated adjusted
odds ratios (AORs) of conception in the FUMC using logistic regression models. Many variables were screened for confounding. AORs for
conception in the FUMC were consistently reduced only for couples
exposed in the second month before conception to [SO.sub.2] levels as
follows: 40-80 [micro]g/[m.sup.3], AOR 0.57 [95% confidence interval (CI), 0.37-0.88; p [is less than] 0.011]; [is greater than or equal to]
80 [micro]g/[m.sup.3], AOR 0.49 (CI, 0.29-0.81; p [is less than] 0.006).
The association was weaker in the second 2 years of the study, probably
due to the gradual decrease of [SO.sub.2] levels in the region. The
relationship between [SO.sub.2] and fecundability was greater in couples
living close to the central monitoring station (within 3.5 km). The
timing of these effects is consistent with the period of sperm
maturation. This is in agreement with recent findings; sperm
abnormalities originating during spermatid maturation were found in
young men from Teplice region who were exposed to the increased levels
of ambient [SO.sub.2]. Alternative explanations of our results are also
possible. Key words: air pollution, environmental exposure,
fecundability, human fertility, reproductive effects, [SO.sub.2], sperm
maturation. Environ Health Perspect 108:647-654 (2000). [Online 5 June
2000]

Several recently published papers have suggested that air pollution
has detrimental effects on reproduction (1-5). Reduced birth rates in
periods with high sulfur dioxide levels were found in a heavily polluted
region of Northern Bohemia during the late 1980s (6,7). Sram and
colleagues (6,7) hypothesized that [SO.sub.2] or some associated
copollutants may reduce reproductive success through adverse effects on
oocyte fertilization, an effect that had been produced experimentally
(8). Detailed information on the course and outcome of almost all
pregnancies occurring in the same region of the Czech Republic was
collected over the last 5 years; relevant pollution data were also
obtained. The purpose of the present paper is to verify the impact of
[SO.sub.2] on fertility in the population.

The probability of conceiving during the menstrual cycle--fecundability--varies considerably, even for healthy and
reasonably sexually active pairs in the human population. Some
proportion of couples will conceive in the first unprotected menstrual
cycle (FUMC), but others need more time to become pregnant. Little is
known about the particular causes of these differences (9). Obviously,
this variation is mostly due to the biologic and social differences
among parental pairs (10); however, fecundability may be also influenced
by environmental factors such as temperature, photoperiodicity, and food
availability or quality (11). There is even growing evidence that some
occupational (12-14) and lifestyle factors (15,16) and some
environmental noxae can adversely affect human fertility (7,17).

Exogenous factors may influence the reproductive ability of
parental pairs by affecting different functions in various levels and
stages of the reproductive process (17); biologic as well as behavioral
functions of one or both partners may be impaired. Couples exposed to
adverse factors take longer time to achieve a clinically recognizable
pregnancy. Therefore, the common consequence of such
effects--irrespective of the particular mechanism--may be a conception
delay. Thus, fecundability can be simply measured as the number of
nonprotected menstrual cycles (MCs) required to become pregnant. This
approach called time to pregnancy (TTP) was introduced in the late 1980s
(13,18) and it has been useful in numerous epidemiologic studies (19-23).

A conception is usually not recognized before the 5th week of
gestation, and only approximately two-thirds of pregnancies reach this
stage (24,25). Data about TTP obtained from parents can be related only
to recognized pregnancies. These data reflect not only the effects of
factors that reduce the probability of conception itself (interfering
with gametogenesis, transport of gametes, or their fertilization
ability); they also include the effects of losses of conceived fetuses
(loss during transport of zygotes, failure to successfully implant, and
especially subclinical abortion).

The TTP approach does not allow for differentiating among these
mechanisms. However, it may be sensitive enough to determine relatively
weak effects on human fertility that could be expected for such common
toxins as air pollutants. Thus, we applied a modified version of the TTP
method in the present study to investigate the impact of [SO.sub.2] on
human fertility.

Materials and Methods

The present study was designed as a prevalence population study
with interviews at delivery. The background sample included all
full-term singleton births in the district of Teplice between April 1994
and March 1998. We excluded all couples who admitted that they "did
something to prevent the index pregnancy" or they "were
treated for fertility disorders." Only spouses of European origin
were enrolled to avoid additional variability related to ethnic and
cultural differences. The sample was further restricted to the
mother's first delivery during the study period. The completion of
a written informed consent form was the final condition for enrollment
in the study.

Personal and lifestyle data were obtained via questionnaires and
medical records. Self-administered maternal questionnaires were
completed in the hospital after delivery, with the assistance of a
specially trained nurse. These data included occupational and other
risks, smoking, and consumption of alcohol, health status and medication
and detailed information on reproductive history and habits. The main
questions concerning TTP from the standard short questionnaire
recommended by Baird et al. (26) were included:

1) Were you (or your partner) doing something to prevent pregnancy at the
time you most recently got pregnant? If no: 2) Did you get pregnant during
the first menstrual cycle of unprotected intercourse? 3) the second 4) the
third 5) other? Please specify which cycle ...

Mothers who answered "yes" to question 1 were excluded
from the study.

Because the fathers were not interviewed, the women were proxy
reporters for their partners' information; more data are therefore
missing for paternal characteristics. Hospital staff abstracted medical
and health care data on the course and outcome of the index pregnancy
from the clinical records.

We estimated the gestational age in weeks using each woman's
prenatal history log (her maternity card), which included her reported
last menstrual period (LMP), plus data on prenatal visits, ultrasound
measurements, etc. We calculated the estimated date of conception (EDC)
using the gestational age and correcting from the LMP (decreasing the
gestational age by 2 weeks) (5).

In the original version of the TTP method, an effect on
fecundability is measured by comparing the distribution in exposed and
nonexposed couples of the number of menstrual cycles required to become
pregnant. In this arrangement, a single exposure estimate is not
meaningful for permanently changing exposures such as air pollution. On
the other hand, the exposure and its timing can be easily defined for
parents who become pregnant in the FUMC. Moreover, according to Baird et
al. (9), the proportion conceiving in the first cycle gives an unbiased
estimate of the mean fecundability in the cohort.

Based on these considerations, we developed a simplified version of
the TTP method: only couples achieving a clinically recognized pregnancy
in the FUMC were categorized as successfully "conceived";
others were classified as "nonconceived." This approach
examines whether the proportion of women who become pregnant in the FUMC
may differ according to the exposure of the parents to air pollution
before conception. We estimated the exposure of each parental pair using
mean 30-day averages of [SO.sub.2] levels in each of 4 months before the
EDC. We used three empirically chosen concentration intervals for
[SO.sub.2] according to exposure distribution in population (low, [is
less than] 40 [micro]g/[m.sup.3]; medium, 40 to [is greater than] 80
[micro]g/[m.sup.3]; and high, [is greater than or equal to] 80
[micro]g/[m.sup.3]). The cutoffs were near tertiles in the first 2 years
of study (27); they fell to 55, 25, and 20%, respectively, during the
last 2 years as a consequence of the downgrading of [SO.sub.2] in
ambient air. We sorted parental pairs by exposure to [SO.sub.2] levels
in the particular month before pregnancy and by conception success in
the FUMC. Conception rates in the FUMC were estimated for each
concentration interval and each month before pregnancy.

Centralized air pollution monitoring in the Teplice district was
organized in cooperation with the U.S. Environmental Protection Agency (U.S. EPA) (28). The monitoring station was located in the center of
town of Teplice. Concentrations of [SO.sub.2] were measured continuously
by the pulse fluorescence method using model 43A (Thermo Environmental
Instruments, Inc., Franklin, MA). Several other pollutants were also
measured, including nitrogen oxide, particulate matter, and polycyclic
aromatic hydrocarbons.

Individual exposures to [SO.sub.2] were derived from centralized
monitoring of ambient air. To reduce inevitable misclassification and
improve the exposure estimate, we used some additional variables. A
unified system of protection for inhabitants has been in place in the
Teplice District since 1993. In periods with extremely high air
pollution levels (for example, meteorologic inversion) district
authorities broadcast a special signal via local media and establish a
special phone service [3-hr average [SO.sub.2] [is greater than] 400
[micro]g/[m.sup.3] or the sum of 24-hr average [SO.sub.2] + (2 times
total suspended particles) [is greater than] 750 [micro]g/[m.sup.3]].
Inhabitants are encouraged to reduce outdoor activities. They are also
encouraged to delay or reduce airing of their homes. Information about
the timing of such signals was obtained from district authorities. A
variable signal was defined as "yes" for each 30-day period
that included a high-pollution episode, under the assumption that
behavior would be altered during those times, resulting in a reduction
of personal exposure.

Two other approaches were applied to take into account the
influence of nonstandard behavior of inhabitants during inversions: we
excluded all parental pairs who were considered exposed to extreme
levels of pollution before conception. Two versions of this sample
restriction were a) the exclusion of couples who were considered to be
exposed to monthly mean concentrations [is greater than] 110
[micro]g/[m.sup.3], which were observed only in rare inversion episodes,
and b) the exclusion of couples who were exposed to inversion situations
during the 4 months before conception.

We expected that the authenticity of the exposure estimates would
be higher for couples living nearer to the monitor irrespective of the
distances of sources or the wind direction. To test this presupposition,
we analyzed the relationship between estimated exposure to pollution and
fecundability separately for parents living at different distances from
the centralized monitor. For this purpose, parental pairs were
classified into two groups (in and out) according to the distance of
maternal permanent residence from the monitoring station in the year
before delivery. Couples living within 3.5 km of the monitoring station
were classified as in; all others were classified as out (Figure 1).

[Figure 1 ILLUSTRATION OMITTED]

Many characteristics are related to human fertility (17,26) and to
[SO.sub.2] exposure. We initially examined the relationships between
fecundability in the FUMC and the characteristics of the parents using
t-test and chi-square analyses. These results were used to construct
logistic regression models.

We estimated adjusted odds ratios (AORs) and their confidence
intervals (CIs) and Wald's chi-squares using logistic regression
procedures (29). Conception in the FUMC resulting in a live birth was
entered as the outcome measure in logistic regression models. A spectrum
of parental characteristics associated with the outcome (fertility) and
some parameters associated with the exposure (pollution) were tested for
inclusion in the final model. These characteristics were maternal age ([is less than] 19, 19-34, [is greater than or equal to] 34 years),
maternal and paternal education (basic, high school, university),
marital status (currently married, other), parity (first, second or
third, higher), spontaneous abortion ([is less than] 2, [is greater
than] 1), induced abortion ([is less than] 2, [is greater than] 1),
alcohol habits of mother and father ([is less than] 1 drinks in a week,
other), maternal smoking before conception (0, 1-9, [is greater than]
9/day) passive smoking (0, 1-9, [is greater than] 9/day) and paternal
smoking (0, 1-9, [is greater than] 9/day), employment of mother and
father (employed yes or no), and occupational risk of mother and father
(yes or no). Eight types of occupational risk (as radiation, chemicals,
dust, infection, etc.) were screened in the models.

A possible confounding effect due to other pollutants was also
taken into account. The findings of several preliminary studies showed
that simultaneous inclusion of highly correlated variables in one model
gives rather misleading results (27,30). The mutual correlations between
levels of [SO.sub.2] and such pollutants as particulate matter or
[NO.sub.x] were high in both regions studied; therefore, we did not
include other pollutants in the models. Although pollutant levels were
associated with season, season by itself might also be a surrogate for
other changes such as weather patterns and the consumption of fruits and
vegetables. We defined summer as the months from April through September
and winter as the months from October through March. We also tested
other definitions of the seasons using four 3-month annual periods.

Secular and/or seasonal rhythms of conceptions in Czech population
were estimated on the basis of 10-year data from official statistics
(31); a parameter rhythm was introduced, weighing each calendar month by
its relative contribution in long-time year totals.

Temperature is an important factor that may influence fertility
(11). For that reason, we introduced temperature in logistic models as
expressed in four variables: avg (monthly average temperature),
[avg.sub.max] (monthly average of daily maximum temperatures), max
(dummy variable introduced for months that included [is greater than] 10
days that were warmer than 80% of the days in a year), and [avg.sub.min]
(monthly average of daily minimum temperatures). Common respiratiory
infections such as influenza are more frequent in the winter. The sexual
behavior of couples (coital frequency) may change during such periods.
We defined a weekly incidence above 1,500/100,000 of acute respiratory
diseases in the Teplice District as an epidemic situation. Using a dummy
variable epidemic situation, couples were differentiated according to
their exposure to this situation during the particular month before
conception; we tested the variable for inclusion into logistic models.

Because air pollution levels have changed in Teplice District over
time, the potential for secular changes was evaluated by comparing
results from the two 2-year periods (period I, from April 1994 to March
1996 and period II, from April 1996 to April 1998).

Each month before pregnancy was analyzed separately, allowing some
factors to vary over time (e.g., pollution levels, temperature, and
season). Some factors considered for analysis were highly correlated
(e.g., mother's active and passive smoking and paternal smoking;
temperature variables; season and conception rhythms). Therefore,
development of the final model used a stepwise approach to select the
most appropriate factors. After this initial stage, we examined other
factors for potential confounding by examining AORs of [SO.sub.2]
exposures to see how much change results from inclusion versus exclusion
of the potential confounder. Change by [is greater than] 15% ([is
greater than] 0.15) for beta was used as a reasonable criterion for
inclusion (29). We used SAS program 6.12 for statistical analysis (32).

Results

There were 3,651 singleton live births to parental pairs between
April 1994 and March 1998 in Teplice District. More than one quarter of
the parental pairs [964 (26.4%)] admitted they did something to prevent
the index pregnancy and were therefore excluded from the study; another
102 were excluded because of previous consultation or treatment for
infertility. Of the 2,585 parental pairs included, 587 (22.7%) conceived
in the FUMC.

Monthly mean levels of [SO.sub.2] during the 4 years of the study
are shown in Figure 2. Average concentrations were generally lowest
during summer and highest during winter. Annual mean levels of
[SO.sub.2] decreased consecutively during the 4 years of the study:
1994-1995 = 54.3 [micro]g/[m.sup.3], 1995-1996 = 50.5
[micro]g/[m.sup.3], 1996-1997 = 48.9 [micro]g/[m.sup.3], and 1997-1998 =
38.0 [micro]g/[m.sup.3]. The overall decrease would have been greater
had it not been for an inversion episode (20 days of extreme [SO.sub.2]
levels from 20 December 1996 through 20 January 1997) (Figure 2). The
mean levels of [SO.sub.2] in 2-year periods decreased from 53.6 [+ or -]
27.6 [micro]g/[m.sup.3] in period I to 44.6 [+ or -] 35.7
[micro]g/[m.sup.3] in period II.

[Figure 2 ILLUSTRATION OMITTED]

Results of descriptive analysis are presented in Table 1. They tend
to suggest that only a few characteristics of parents who conceived in
the FUMC differ significantly from those of less successful pairs.
Couples who became pregnant in the FUMC were significantly more
frequently single (32.2 vs. 26.7%; p [is less than] 0.01) and conceived
more often during the summer months (55.2 vs. 50.2%; p [is less than]
0.05) compared to the others. Mothers from successful pairs were more
frequently unemployed or employed without occupational risk.

(a) Ambient levels of pollutants during the second month before
conception.

(b) Basic education = approximately 9 years.

(c) High school (with maturity exam) = approximately 11-12 years in
the Czech Republic during the relevant period.

(*) p < 0.05.

(**) p < 0.01.

Table 2 presents the results of multivariate analysis of the impact
of [SO.sub.2] exposure in different months before conception on
fecundability in the FUMC. AORs for conception in the FUMC were
consistently reduced, with higher levels of [SO.sub.2] in the second
month (30-60 days) before conception. The AOR for the medium [SO.sub.2]
level was 0.57 (CI, 0.37-0.88; p [is less than] 0.011); it was 0.49 (CI
0.29-0.81; p [is less than] 0.006) for the high level. No significant
association was observed in any other period before conception (Table
2).

Table 2. AORs of the fecundability in the FUMC by exposure to
[SO.sup.2] before conception.

We conducted a similar analysis for those parental pairs delivering
in the first and the second 2-year periods (n = 1,527 and 1,058 couples,
respectively). The only clear-cut relationship between fecundability and
[SO.sub.2] was observed for pairs delivering in period I in the second
month before conception: for medium [SO.sub.2] levels the AOR = 0.49
(CI, 0.25-0.96; p [is less than] 0.037) and for high levels the AOR =
0.43 (CI, 0.20-0.93; p [is less than] 0.033). In period II there was a
consistent but nonsignificant tendency (Table 3).

Table 3. AORs of the fecundability in the FUMC by exposure to
[SO.sub.2] before conception,

The variable signal was associated with [SO.sub.2] exposure.
Inclusion of this covariate into logistic models tended to decrease
slightly the AORs for the fourth and first-months before conception; we
did not see an influence on the AORs for the third and second
preconceptional months. Another way to reduce possible distortion of the
association between exposure and effect during inversion episodes was to
exclude cases exposed to inversion from the sample. After the exclusion
of 63 parental pairs exposed to monthly means [is greater than] 110
[micro]g/[m.sup.3] from the whole sample, the AORs for both medium and
high [SO.sub.2] levels were further reduced (0.51 and 0.42 instead of
0.57 and 0.49), and significance levels were higher (Table 4). This
approach was also applied to the analysis of samples for periods I and
II. The exclusion of exposures [is greater than] 110 [micro]g/[m.sup.3]
did not change the results in period I. In contrast, the results for
period II change considerably when high exposures are excluded (Tables 3
and 4): the AOR for medium exposures was 0.54 (CI, 0.28-1.07; p [is less
than] 0.08) and the AOR for high exposures was 0.44 (CI, 0.18-1.09; p
[is less than] 0.08). Thus, after exclusion of extremely high exposures,
the results in the second 2 years of the study are similar to those from
the first 2 years, though with the marginal significance only. The
results of the alternative analysis of the samples after exclusion of
the 92 couples exposed to inversions before conception yielded similar
results (Table 4). Approximately one-half of the couples (1,297) lived
up to 3.5 km away from the central monitor (classified as
"in") (Figure 1). Results in Table 5 show that the association
between conception success in the FUMC and [SO.sub.2] exposure in the
second preconception month is greater for the group who lived closer to
monitor ("in"). In the "in" group, the AOR of
conception in the FUMC was consistently decreased for medium (0.56; CI,
0.31-1.00) and high (0.36; CI, 0.17-0.73) [SO.sub.2] exposure. The
fecundability/[SO.sub.2] association in the "out" group was
weaker and nonsignificant (Table 5). Excluding both the "out"
group and the inversion cases magnified even more the effect of
[SO.sub.2] on fecundability in the second month before conception (Table
5).

Table 4. Effect of exclusion of extremely high exposures to
[SO.sub.2] in the second month before conception.

(d)n = 2,522. All mean month [SO.sub.2] exposures > 110
[micro]g/[m.sup.3] occurred during the inversion episode from 15
December 1996 through 20 January 1997; thus, only results for the second
2-year period II (April 1996 to March 1998) were influenced.

(e) n = 2,493. Results of analysis after exclusion of couples
exposed to the inversion situation did not differ from those calculated
after exclusion of extremely exposed parents. Either extremely high
exposures or altered behavior of people during the inversion episode 15
December 1996 through 20 January 1997 might distort the
[SO.sub.2]/fecundability detected in this study.

Table 5. Influence of the distance from the monitor on AOR of
fecundability in the second month before conception.

(c) Combined effect of the limitation of the distance of residence
from the monitoring station (< 3.5 km) and the limitation of
considered exposure to [SO.sub.2] (< 110 1 [micro]g/[m.sup.3]).

Discussion

Teplice District lies in a highly industrialized mining area of
Northern Bohemia with heavy industry and many large power plants using
low-energy-quality brown coal with high sulfur content. Pollution
reaches its highest levels during meteorologic inversions, which are not
infrequent events in this mountainous area. Ambient air monitoring tends
to suggest that [SO.sub.2] levels were falling during the 4 years of
this study. This trend seems mainly to be due to changes in industry
profiles, technological improvement of large power plants, and a rapid
conversion of local heating systems from coal to gas in the Teplice
area.

The longitudinal version of the TTP method presents a problem for
evaluating the impact of air pollution on fecundability. All potential
parents were continually exposed in various periods before a particular
conception and the levels of pollutants are continually changing.
Appropriate comparisons for couples conceiving in the FUMC are easier to
achieve. Baird et al. (9) suggested that data about the proportion
conceiving in the first cycle give an unbiased estimate of the mean
fecundability in the cohort, providing a rationale for the design of the
present study. In this approach, information concerning the distribution
of later conceptions is lost. On the other hand, a bivariate outcome
measure and yes or no responses make it possible to apply logistic
regression (9), a powerful device for controlling potentially
confounding covariates. It should be emphasized that the approach used
is based on data obtained at delivery; therefore, all early losses were
included within the nonconceived group; on the other hand, all
conceptions that resulted in clinical spontaneous or induced abortion
were omitted.

We used the EDC as a reference date for analysis. Stolwijk et al.
(33) recently showed that this approach might involve substantial bias
arising from a seasonal pattern of pregnancy planning. The authors
instead recommended using the date of onset of TTP, which is not biased
in this manner. This recommendation cannot be followed in the present
study, as data about TTP onset cannot be obtained from couples with
longer conception delay. Therefore, we included annual conception
rhythms observed longitudinally in the Czech population in logistic
models; thus a possible influence of seasonality in pregnancy planning
was reduced. According to Stolwijk et al. (33), a residual effect of
this bias can cause an underestimation of the strength of the relation,
but not a change in the direction of the effect estimators.

Results of descriptive analysis suggested that parental pairs
conceiving in the FUMC did not differ from less successful couples in
most of the characteristics listed in Table 1. Mothers conceiving in the
FUMC were more frequently single and unemployed. The employed women from
this group were less likely to be exposed to occupational risk. After
the inclusion of particular occupational risks into logistic models, the
AOR of conception in the FUMC was significantly reduced for mothers
exposed to any risk (0.75; CI, 0.58-0.97), and to radiation (0.30; CI,
0.69-1.12); the same is true for fathers exposed to dust (0.64; CI,
0.46-0.89). Inclusion of these variables in the models did not influence
the final association between [SO.sub.2] and fecundability in the FUMC.
Success in the FUMC is more frequent during the summer (24.4%) than
during the winter (20.9%). This could be due to better opportunities for
intercourse during vacation time and to other supporting influences of
summer (10). Moreover, couples successful in the FUMC were exposed to
lower levels of [SO.sub.2] during the second month before conception
(Table 1). Thus, association between [SO.sub.2] and fecundability may
also contribute to higher conception success in the FUMC during summer.

We used monthly means of [SO.sub.2] to characterize the exposure.
We examined shorter as well as longer periods in preliminary
investigations to find an optimal measure. Misclassification of exposure
estimates may be frequent, and the accuracy of the EDC is limited in the
present study. An interval shorter than 30 days is inadequate to ensure
the reliability of data. On the other hand, the biologic sensitivity
window (e.g., in the case of influence on spermatogenesis) can be
relatively narrow, on the order of weeks. To hit this small target using
such an inaccurate device requires choosing the optimal interval, such
as 1 month, because only analyses based on 30-day periods yielded
consistent results.

Ambient [SO.sub.2] is only one of many components of a complex
mixture. The possible effects of [SO.sub.2] and four other noxae, namely
[NO.sub.x], particulate matter [is less than or equal to] 10 [micro]m in
aerodynamic diameter ([PM.sub.10]), particulate matter [is less than or
equal to] 2.5 [micro]m in aerodynamic diameter, and polycyclic aromatic
hydrocarbons, on fecundability were examined in two preliminary studies
using the same approach (27,30). Levels of all five noxae were mutually
highly correlated in a range of 0.55-0.83. Analyzing each pollutant in a
separate model, we observed the only consistent relationship to
fecundability was that for [SO.sub.2]. A much weaker association with
[PM.sub.10] could be explained by the high correlation between
[SO.sub.2] and [PM.sub.10] levels (r = 0.83; p [is less than] 0.0001).
An analysis of the simultaneous effects of the two pollutants in one
model yielded an increased [SO.sub.2] effect (AORs for [SO.sub.2] 40-80
[micro]/[m.sup.3] = 0.53; CI, 0.39-81 and AORs for [SO.sub.2] [is
greater than or equal to] 80 [micro]g/[m.sup.3] = 0.41; CI, 0.25-0.70)
and eliminated any suggestion of [PM.sub.10] association (AORs for
[PM.sub.10] 40-50 [micro]g/[m.sup.3] = 1.18; CI 0.90-1.56 and AORs for
[PM.sub.10] [is greater than or equal to] 50 [micro]g/[m.sup.3] = 1.30;
CI, 0.92-1.93) (30). These results would be misleading in view of the
high correlation of both variables. Therefore, the analysis was
concentrated on the effect of [SO.sub.2] alone in the present
communication. The rather complex question of the simultaneous effects
of copollutants will be discussed in a separate study.

Multivariate analysis of data from all 4 years showed a relatively
strong inverse association between the concentration of [SO.sub.2]
during the second month before conception and conception success in the
FUMC (Table 2). The AOR of conception in the FUMC was reduced to 0.57
for couples exposed to [SO.sub.2] levels of 40-80 [micro]g/[m.sup.3] in
the second month before conception. This value falls to 0.49 for those
exposed to levels [is greater than] 80 [micro]g/[m.sup.3] in the same
preconceptional stage. A similar relationship was also observed when
data were divided into two subsets corresponding to the first (period I)
and second (period II) 2-year periods of the study (Table 3). However,
the results show unequivocally that the association is much stronger in
period I. The particular AOR values were 0.49 and 0.43 for medium and
high [SO.sub.2] exposures; lower than for the total 4-year sample (Table
2). On the other hand, the association in period II was much weaker; in
fact, it was no longer significant. This tendency may be rooted in the
permanent decline of [SO.sub.2] levels in the region during the past
several years.

Misclassification in exposure estimates is a usual weakness in
studies based on centralized monitoring. To reduce the deleterious effects of misclassification, we conducted a sensitivity analysis. The
variable signal should correct possible exposure-protective behaviors of
people to the warning system during inversion episodes. However, an
introduction of this variable into logistic models did not affect the
results in any preconception period. Another method used to control the
influence of possible protective behavior of inhabitants during
inversion episodes was the exclusion of inversion-exposed cases from the
analysis. This approach seemed to clarify the exposure/outcome
associations. An exclusion of parental pairs exposed to monthly means of
[SO.sub.2] [is greater than] 110 [micro]g/[m.sup.3] (63 couples) had the
same effect as the exclusion of 92 couples who were exposed to an
inversion episode before pregnancy (Table 4). The reduction of AORs for
medium and high exposures in the second month before conception was
stronger and more significant. It may be particularly important that the
difference between the effect of [SO.sub.2] on fecundability in periods
I and II was reduced after controlling for the influence of inversion
situations.

We derived the mean monthly exposure of couples to [SO.sub.2] from
daily measurements by a central monitor. Exposure estimates should be
more accurate for persons living near the monitoring station than for
others, irrespective of the distance of pollution sources or wind
direction. If the relationship between [SO.sub.2] and fertility is real,
the statistical association between estimated levels of [SO.sub.2] and
fecundability derived from logistic models should be stronger for the
"in" sample. The present results show unequivocally that the
relationship between [SO.sub.2] and fertility for couples living [is
less than] 3.5 km from the monitor station is much stronger than for
other pairs (Table 5). Approximately one-half of the inhabitants of the
district were living inside and the other half outside this area (1,297
and 1,288, respectively): this made the cutoff optimal for comparison of
both subsamples. This result strengthens the hypothesis that some part
of the variation in fecundability may be explained due to the changes in
[SO.sub.2] levels.

It has been suggested that temperature may affect human fertility
(11). Temperature may influence hormonal levels (34), frequency of
intercourse (11), and spermatogenesis (35). In our study, average as
well as maximal temperatures in the second preconceptional month showed
significant association with fecundability in the FUMC in models without
pollutants (p [is less than] 0.003 and p [is less than] 0.03); no
consistent association of fecundability with temperature during other
months was observed. In models containing [SO.sub.2], both maximal and
average temperatures significantly influenced AORs, especially in the
second month before conception. On the other hand, the influence of
[SO.sub.2] in models without temperature reduced the fecundability
significantly although the AORs were lower than in the complete model
[medium [SO.sub.2] level AOR = 0.67 (CI, 0.44-0.96; p [is less than]
0.03); high [SO.sub.2] level AOR = 0.60 (CI, 0.38-0.97; p [is less than]
0.03)]. Minimal temperatures did not affect the [SO.sub.2]/fecundability
association and were not included into final models. Success in the FUMC
was observed significantly more frequently in the summer (from April
through September) than in the winter (Table 1). Conception rates in the
FUMC were also positively related to the warmest periods in the logistic
models used. These observations are not surprising because the
conditions for fertilization are generally more favorable during the
warmer months in temperate latitudes (36). Loose summer clothing enables
better scrotal cooling, which optimizes spermatogenesis and sperm
quality. Other influences such as higher coital frequency in the summer
can also contribute to this seasonal fluctuation (37). It seems that
seasonal epidemics of respiratory diseases may influence fecundability.
The observed fecundability/[SO.sub.2] association in the second month
before conception decreased slightly after controlling for the influence
of epidemic situations. On the other hand, AORs for other periods before
conception tended to decline to unity as if those epidemics may explain
a part of the variability in fertility. Some changes in sexual behavior
during such epidemics can be hypothesized. Thus, fecundability may vary
due to many seasonal factors other than pollution.

Currently, few papers have examined possible associations between
human fertility and air pollution. Recently, reduced birth rates were
observed during periods of high [SO.sub.2] concentrations in the Teplice
region (6,7). It has been suggested that high [SO.sub.2] or some
associated pollutants may reduce the ability of oocytes to be
fertilized. Jagiello (8) previously observed this effect experimentally.
Sram et al. (7) hypothesized that an effect of other environmental
mutagens, inducing mutations in gametes or early embryos, may be
potentiated by [SO.sub.2] due to the suppression of DNA repair mechanisms.

Whether [SO.sub.2] affects fertility through genetic mechanisms
remains an open question. There are conflicting data about the genotoxic effects of [SO.sub.2] in humans. Schneider and Calkins (38), Nordensen
et al. (39) and Yadav and Kaushik (40) observed clastogenic effects in
workers exposed to high concentrations of [SO.sub.2]. Chromosomal
aberrations and sister chromatid exchanges (SCEs) in exposed workers
were observed by Meng and Zhang (41). On the other hand, Sorsa et al.
(42) observed neither chromosomal aberrations nor SCEs.

Little is known about the background variability in fecundability
in the normal human population (10,17,26). It is clear that delays in
conception may result from a spectrum of pathogenetic processes in one
or both sexes (17). The present approach cannot differentiate among the
processes involved; therefore, we can only speculate about mechanisms of
the observed adverse effects of [SO.sub.2]. However, the timing of the
acute effect coincides with some important stages of the reproductive
process in men, namely sperm maturation. This finding is interesting in
light of the recent results of Selevan et al. (43), in which semen
quality was repeatedly analyzed in healthy young men from Teplice.
Highly significant adverse but transient effects of increased [SO.sub.2]
levels on sperm morphology and motility during one spermatogenic cycle
were observed in multivariate analysis. The results tended to suggest an
effect on spermatogenesis rather than an acute influence on epididymal sperm function. These effects should operate during transformation of
the round spermatids into differentiated sperm cells (43). These results
support our findings with regard to the timing of conception: it is
noteworthy that in our results a significant reduction of the conception
rate in the FUMC was associated with [SO.sub.2] levels only in the
second month before fertilization. This is the same period described by
Selevan et al. (43) as the most probable stage of sperm maturation
damage associated with [SO.sub.2] exposure. In addition, these
authors' results contribute to the substantive question of the
relationship between [SO.sub.2] and fertility: there are indications
that the type of sperm damage observed by Selevan et al. (43) may
actually reduce fertility (44).

In spite of this late preconception period, an interference of
[SO.sub.2] or some copollutant with the respective stages of oogenesis
cannot be excluded. Damage to gametes might reduce the efficiency of
reproduction, subsequently affecting fertilization, implantation, and
early embryogenesis. All of those events could increase the risk of
early losses (subclinical abortion). On the other hand, toxic pollutants
may also interfere with these processes directly.

We are aware that our results are based on some data of unequal
reliability. Some questionnaire data (parental ages, date of delivery,
and other personal data) were crosschecked using other information
sources. Pollution data were measured using the regularly calibrated equipment and standardized methods developed by the U.S. EPA; these data
should be reliable. However, we made an important assumption in the
assignment of exposure to different time periods: the measured exposure
level at the central monitoring station may not be entirely
representative of an individual woman's or man's exposure.
Wide variations in exposure may result from individuals living at
various distances from the monitor, varying wind conditions, varying
personal habits, and differences in daily routine. Exposure estimates
were more relevant for couples living near the monitoring station (Table
4). The exposure-effect relationship may be distorted in periods with
extremely high levels of pollution (inversions).

Another possible source of error in exposure estimation may be
incorrect determination of the EDC. Errors in EDC could blur or wholly
dissolve the exposure-period relationship; systematic error may shift
the important exposure to another time period. To prevent this, we made
the EDC determination using maternal prenatal records obtained in early
pregnancy. Thus, systematic errors in the EDC are less likely.

Conclusions

The results of the present study suggest that AORs of conception in
the FUMC may be reduced in couples exposed to mean [SO.sub.2] levels [is
greater than] 40 [micro]g/[m.sup.3] in the second month before
conception. No consistent relationship was observed in any other period
during the 4 months before conception. The exposure-effect association
tends to be strengthened by the exclusion of couples living larger
distances from the monitor station and/or couples exposed to extreme
inversion situations, when behavior may alter exposures. The timing of
the effect coincides with the sperm maturation period. These results are
in agreement with the findings of Selevan et al. (43), who observed
spermatogenesis damage in the same stage in young healthy men exposed to
ambient [SO.sub.2]; the observed types of damage may reduce fertility.

The impact of preconceptional exposure to other pollutants will be
evaluated in the future using the same methods.

We thank I. Hertz-Picciotto for invaluable comments and critical
review of the manuscript. We thank the many gynecologists and their
staff members from the Departments of Obstetrics and Gynecology in
hospitals in Teplice and Duchcov for their excellent collaboration. We
also thank our colleagues from the District Institutes of Hygiene in
Teplice and Prachatice for their support and collaboration.

Supported by grants from the Czech Ministry of Environment (Teplice
Program II), U.S. Environmental Protection Agency/U.S. Agency for
International Development, and CEC (PHARE II, EC/HEA-18/CZ).

Received 3 December 1999; accepted 14 March 2000.

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